The invention described herein relates generally to structures and methods for performing fluorescence analyses of single particles and assemblies of particles, and more particularly to apparatus and methods for simultaneously measuring fluorescence intensities at a plurality of excitation and emission wavelengths, and at a plurality of fluorescence lifetimes of single particles and assemblies of particles both locally and remotely.
The ability to extract fluorescence signals from a background on the basis of specific excitation, emission and lifetime properties of fluorochromes is essential in such diverse fields as cytometry and lidar-based remote detection. The invention presented in this disclosure concerns a method and the corresponding apparatus for extracting specific fluorescence signals in real time from a light background, the extraction process simultaneously making use of specific excitation, emission and lifetime properties of the fluorochromes.
Much flow cytometric and imaging work in biology is concerned with the quantitation of probes that bind in a specific manner to various biological structures. Such probes are usually fluorescent (either intrinsically or through the chemical attachment of fluorescent molecules), and can thus be detected by flow and imaging analytical instruments through optical and photometric means.
It is preferable in many such applications to use several, simultaneous probes. The simultaneous presence of a range of probes of different specificities may reveal, if the probes can be independently quantitated, a correlated picture of the distribution of several biochemical determinants in a cell population (flow cytometry) or inside a cell or tissue (fluorescence microscopy). If the probes are fluorescent and the fluorescence of each probe has distinctive properties (in terms of its excitation and emission spectra, as well as of its fluorescence lifetime spectrum), then these properties may be used by an analytical instrument in order to separate the contributions of the different probes to the total fluorescence, and thus independently quantitate the various probes.
Previous approaches to the problem of fluorochrome separation have used different optical and data-processing techniques for each set of fluorescence properties (excitation, emission, and lifetime). Of these, the techniques for measuring excitation and emission properties are, however, similar. In general, they are based on the spatial or temporal division of the excitation and emission spectra into a number of spectral channels, the measurement of the fluorescence intensity being correlated in space and/or time with the excitation and emission spectral channels. For example, in fluorescence microscopy, there may be several excitation and emission filters mounted on filter wheels, with fluorescence intensity measurement being successively made for various combinations of filters. In flow cytometers, different excitation wavelengths may be provided at different locations along the particle-carrying stream by different laser beams, thus providing a temporal sequence of excitation wavelengths for particles in the stream. The emission spectrum is spatially divided into regions of interest by combinations of optical filters and partially reflecting surfaces, with one detector for each spectral region.
Measurements of fluorescence lifetime are usually based on modulation techniques, whereby the intensity of the excitation light is temporally modulated according to some modulation scheme (harmonic, multiharmonic, periodic sequence of narrow pulses), and the lifetime information is extracted from the shape of the emission intensity waveform. A lifetime measurement in flow has yet to be demonstrated. In imaging systems, measurements have been performed for long-lived, delayed fluorescence or phosphorescence.
Another field in which a fluorescence signal must be extracted from a background is that of fluorescence lidar. For example, the remote detection and mapping of the distribution of biological aerosols can be achieved by projecting a pulsed laser beam of appropriate wavelength into the atmosphere and detecting the fluorescence emitted by tryptophan, an ubiquitous component of proteins. If the tryptophan fluorescence signal can be extracted from the background light, then the distribution of delays between the excitation laser pulse and the fluorescence signals can be used to map the spatial distribution of the aerosol.
The use of specific excitation and emission properties of fluorochromes to be detected by lidar are useful in extracting their fluorescence signals from the background. The lifetime properties are often more useful, especially if one is measuring the fluorescence intensity time-response function. In this situation, the fluorochrome's characteristic fluorescence decay is shifted in time by an amount equal to the sum of the propagation times for the excitation and fluorescence pulses. Thus, the fluorescence time-response function contains information on both lifetime and distance. The two can be easily separated if the lifetime is much shorter than the propagation delay, which is typically the case in fluorescence lidar applications.
Accordingly, it is an object of the present invention to provide an apparatus and method for allowing the simultaneous measurement of excitation, emission and lifetime properties of fluorochromes using a single, integrated instrument.
Another object of the invention is to provide an apparatus and method for allowing the remote, simultaneous measurement of excitation, emission and lifetime properties, and the spatial distribution of fluorochromes using a single, integrated instrument.
Yet another object of the present invention is to provide an apparatus and method for allowing the simultaneous measurement of excitation, emission and lifetime properties of fluorochromes in single particles using a single, integrated instrument.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.